Purification of a pertussis outer membrane protein

ABSTRACT

Pertactin (formerly 69 kDa protein) is recovered in stable biologically pure form having no detectable adenylate cyclase activity from fermentation broth from the fermentation of Bordetella pertussis as well as from the cells. The broth is processed to selectively remove pertussis toxin (PT) and filamentous haemagglutinin (FHA), the pertactin is precipitated by ammonium sulphate and the precipitate is dissolved in buffer at pH 6.0 to 8.5, the solution then is passed through hydroxyapatite and ion-exchange chromatograph columns before final ultrafiltration. Cells are extracted with urea and the extract ultrafiltered and diafiltered. The pertactin is precipitated from the extract and the precipitate processed as above. In a variation, the broth is contacted with ammonium sulphate to precipitate pertactin, PT and FHA, the precipitate is dissolved and the PT and FHA selectively removed, before the solution is passed to the chromatograph columns.

FIELD OF INVENTION

The present invention relates to a novel process for the purification ofan outer membrane protein of Bordetella pertussis, having a molecularweight of approximately 69,000 Daltons, formerly called the 69 kDaprotein and now called pertactin, and obtained from the fermentationbroth and cellular extracts of the said organism. The protein obtainedby the process is to be used in a "component" vaccine to protect againstthe disease of whooping cough.

BACKGROUND TO THE INVENTION

The disease of whooping cough or pertussis is a result of infection byBordetella pertussis, and is a serious and debilitating human diseaseparticularly in young children. For the last fifty years the disease hasbeen controlled through large-scale immunization programmes. The currentlicensed vaccine in North America is a "whole cell" vaccine prepared bygrowing the organism in fermentors and then treating the resultingB.pertussis cells with chemical agents, such as formaldehyde, to killthe organism and inactivate toxic proteins. The cells are resuspendedand then used directly or in combination with other antigens. Thisvaccine, although highly efficacious, has been associated with clinicalsymptoms that include fever, local reactions, high-pitched crying andconvulsions. Despite the fact that there is no proven relation betweenthese symptoms and the vaccine, there has been decreased publicacceptance of this vaccine and in a number of countries, e.g. Japan,Sweden and the U.K., decreased immunization has led to outbreaks of thedisease. The need for a more defined vaccine has been recognized andconsiderable effort has been directed by several manufacturers andresearchers towards the development of an efficacious pertussis vaccinethat consists of a small number of highly purified proteins. Thisvaccine has been termed a component vaccine.

This search has been hampered by a lack of information on the mechanismof pathogenesis of B.pertussis. Many virulence associated factors, suchas pertussis toxin (PT), also known as lymphocytosis promoting factor(LPF), filamentous haemagglutinin (FHA), adenylate cyclase,lipopolysaccharide, agglutinogens and other outer membrane proteins havebeen suggested for inclusion in an "acellular" vaccine, which is lessdefined than the component vaccine. Much of the work on acellularvaccines has concentrated on a PT-based vaccine. Results of a recentclinical trial have indicated that a vaccine consisting entirely ofPT-toxoid only partially protected children from the infection. A PT/FHAcombination showed slightly higher efficacy but this was still lowerthan that obtained for the whole-cell vaccine.

One potential protective antigen is an outer membrane protein, with amolecular weight of approximately 69,000 daltons (pertactin) found onall virulent strains of B.pertussis. This protein is produced inrelatively large amounts during the culture of the organism and can bepurified from either the fermentation broth or from cell extracts. Thepresent invention provides a novel method of effecting suchpurification.

The potential importance of this outer membrane protein for inclusion ina human vaccine against whooping cough was suggested from attempts toprepare a vaccine to protect pigs against B.bronchiseptica infection.Cell-surface extracts of B.bronchiseptica were used to immunize sows.Levels of antibody to a cell surface antigen with a molecular weight of68,000 daltons correlated with protection of newborn piglets againstinfection. Similar antigens, with similar molecular weights, weredetected in B.pertussis (approximately 69,000 daltons) and inB.parapertussis (approximately 70,000 daltons). Immunization with theprotein obtained from B.pertussis protected mice against intracerebralchallenges with live organisms and antibodies to the protein conferredpassive protection to mice in this test. Both active and passiveprotection of mice in an aerosol challenge model have also beendescribed.

The published procedures for purification of pertactin do not allow forthe large-scale production of a highly purified, non-pyrogenic andstable antigen. One reported method (Canadian Patent No. 1,253,073)involves acid-glycine extraction of the cells, anion-exchangechromatography and preparative iso-electric focussing. However, thepertactin obtained has been reported to degrade into smaller fragments,to be sensitive to low pH and to have adenylate cyclase activity. Forthese reasons, this extraction procedure is considered undesirable forlarge-scale production. In addition, iso-electric focussing is notamenable to large-scale production. A second procedure (U.S. Pat. No.5,101,014) involved the extraction of the outer membrane protein fromthe cells of an afimbriated strain of B.pertussis. The protein waspurified by a combination of DEAE-Sepharose and Affigel-bluechromatographies. The potential of leaching the blue dye into theproduct would be a possible safety concern. Neither method addresses thepurification of pertactin directly from fermentation broths.

SUMMARY OF INVENTION

In one embodiment of the present invention, pertactin is obtained inlarge quantities from fermentor broth, which is the preferred source,and in a purified form, by using the method described below. The proteincan be included in a product to be used for the widespread vaccinationof children against whooping cough.

After growing the organism in a fermentor, the cells are removed bycentrifugation and filtration and the supernatant reduced in volume andsterilised. The broth is diluted to a low ionic strength and, afterremoval of other antigens, the pertactin is isolated by chromatographyon various substrates and further purified by ultrafiltration.

The protein can also be isolated from the cells after extraction withurea, centrifugation and further processing to give a solution that canbe treated as described above.

Accordingly, in one aspect, the present invention provides a method forthe production of pertactin, which comprises providing an impure aqueoussolution of pertactin substantially free from other Bordetella antigens,purifying pertactin in said aqueous solution by passing said aqueoussolution sequentially in contact with hydroxyapatite and an ion-exchangemedium, and subjecting the resulting purified solution toultrafiltration.

The resulting product is very stable when purified by this method andhas no detectable adenylate cyclase activity. Accordingly, anotheraspect of the invention provides a biologically pure and stablepertactin having no detectable adenylate cyclase activity.

GENERAL DESCRIPTION OF INVENTION

The process described in this invention allows for the purification ofseveral protein antigens for possible inclusion in a component pertussisvaccine from a single fermentation of B.pertussis.

In the present invention, B.pertussis is grown in a fermentor undercontrolled conditions. Carbon sources and growth factors aresupplemented either continuously or in batches at various intervalsduring the fermentation until the pertussis proteins (PT, FHA andpertactin) are at the desired levels as determined by a specificenzyme-linked immunosorbent assay (ELISA) for each antigen. Thefermentor broth is harvested, the majority of the cells removed bycentrifugation and the broth sterilised by microfiltration, preferablyusing known membrane filters of about 0.2 micron pore size. The broth isconcentrated, say 10-fold, by membrane ultrafiltration and used for thepurification of pertussis toxin and FHA (see published European PatentApplication No. 0,336,736; U.S. Pat. No. 4,997,915, the disclosure ofwhich is incorporated herein by reference). The cells are the source ofmaterial for the purification of the agglutinogens. Pertactin can bepurified from both the broth or cells. The former is preferred as themajority of the protein is found in the broth.

The first stage in the purification of the pertactin requires dilutionof the broth to a low ionic strength and chromatography on Perlite orother suitable solid particulate adsorbent material to remove the PT andFHA antigens, as more fully described in the aforementioned publishedEuropean Patent Application No. 0,336,736, U.S. Pat. No. 4,997,915). ThePT and FHA antigens can be removed from the adsorbent material bytreatment with an aqueous medium of high ionic strength for use in acomponent pertussis vaccine.

As used herein, the term "low ionic strength" refers to an aqueousmedium having a conductivity of about 11 mS/cm or less, preferably about4 mS/cm or less. The unit of measurement mS/cm is millisiemen percentimeter. A Siemen (S) is a unit of conductivity and is the equivalentof the inverse of resistance (ohm) and is sometimes designated mho. Theterm "high ionic strength" as used herein refers to an aqueous mediumhaving a conductivity of greater than about 11 mS/cm and preferably atleast about 50 mS/cm.

The remaining mixture then is concentrated by membrane filtration,subjected to ammonium sulphate precipitation and the resulting pelletdissolved in low ionic strength buffer, such as Tris-.HCl, at a pH of6.0 to 8.5 to yield a solution with a final conductivity of generallyless than about 4 mS/cm, typically approximately 3.4 mS/cm. The solutionis chromatographed sequentially on hydroxyapatite, and an ion-exchangemedium, such as Q-Sepharose®. Pertactin elutes in the unbound fractionof both columns under the specified buffer conductivity. However, if theconductivity is lower than 1.5 mS/cm for hydroxyapatite or 2.8 mS/cm forQ-Sepharose®, pertactin binds to both columns and can be eluted with abuffer having conductivities 1.5 mS/cm or greater for hydroxyapatite and2.8 mS/cm for or greater Q-Sepharose®.

The pertactin is further purified by ultrafiltration through about 100to 300 kDa Nominal Molecular Weight Limit (NMWL) membranes where it iscollected in the filtrate, concentrated using membranes with a NMWL ofabout 30 kDa or less and sterile filtered for use in combination withother pertussis antigens in a vaccine.

In an alternative procedure, pertactin along with FHA and PT areprecipitated from fermentor broth, by addition of ammonium sulphate. Theprecipitate is removed from the residual broth and redissolved toprovide an aqueous solution suitable for processing as described abovefirst to remove the PT and FHA and then to purify the pertactin.

Pertactin also can be purified from B.pertussis cells. The cells areextracted in a solution containing a high concentration (for example,4M) of urea for, say 1.5 hr. at room temperature. Cell debris is removedby centrifugation and the supernatant, which contains the protein, issubjected to ultra-filtration using 100 to 1000 kDa NMWL membranes. Highmolecular weight proteins, such as the agglutinogens, are retained whilethe majority of the pertactin is filtered through. The filtrate isconcentrated, diafiltered using a 30 kDa or less NMWL membrane, and thenprecipitated by ammonium sulphate and centrifuged to give a pellet forprocessing as described above.

Pertactin, although reportedly susceptible to proteolytic cleavage, isvery stable when purified by the method of the invention. SDS-PAGEanalysis indicates that the purified pertactin is homogenous andessentially intact with traces of degradation products of molecularweights between 30 and 40 kDa. No evidence for further degradation orchange in immunogenicity was found after several months of storage at 2°to 8° C. or at higher temperatures (24° C., 37° C.) In contrast to aprevious publication (Canadian Patent No. 1,253,073), the finding thatthe pertactin prepared by this method does not show any detectableadenylate cyclase activity and has enhanced stability makes it an idealcandidate for inclusion into a component vaccine for protection againstB.pertussis.

EXAMPLES

Methods of protein biochemistry, immunochemistry used but not explicitlydescribed in this disclosure and these Examples, are amply reported inthe scientific literature and are well within the ability of thoseskilled in the art.

EXAMPLE 1

This Example illustrates the growth of B.pertussis in fermentors.

B.pertussis was seeded into a fermentor containing 250 L of broth(modified Stainer-Scholte medium). During the period of fermentation,monosodium glutamate and the growth factors, glutathione, ferroussulphate, calcium chloride, ascorbic acid, niacin and cysteine wereadded during the fermentation process to increase antigen yields. At theend of a 48 hr. fermentation period, the broth was centrifuged to removethe majority of cells and the supernatant, which contains PT, FHA andmost of the pertactin, was further clarified by ultrafiltration throughcellulose acetate membranes (0.22 μm pore size). The sterilised filtratewas concentrated approximately 10-fold using a 20 kDa NMWL membrane andassayed for protein content and for antigen by antigen-specific ELISAs.

EXAMPLE 2

This Example illustrates the large-scale removal of PT and FHA using achromatographic column of Perlite.

The broth concentrate, prepared as described in Example 1, was dilutedwith water to a conductivity of ≦4 mS/cm and subjected to chromatographyon a Perlite column (12 cm[H]×37 cm[D]), previously equilibrated withwater, at a protein to Perlite ratio of approximately 3 mg permilliliter and a linear flow rate of approximately 100 cm/hr. Proteinsbound to the Perlite were almost exclusively PT and FHA while pertactinwas found in the flow-through.

EXAMPLE 3

This Example illustrates the precipitation of pertactin using ammoniumsulphate fractionation.

The flow-through fraction from Example 2 was concentrated to a volume ofapproximately 10 liters by ultrafiltration using 10 kDa NMWL membranes.The resultant solution usually had a protein concentration of 1 to 2mg/ml. While stirring at room temperature, ammonium sulphate (3.5 Kg/10L of concentrate or 35% w/v) was slowly added, and the mixture left todissolve before transferring to a refrigerator at 2° to 8° C. andstirred for an additional two hours, preferably overnight. Theprecipitate was collected by centrifugation and dissolved in 2 liters of10 mM Tris.HCl, pH 6.0 to 8.5. A second ammonium sulphate fractionation(25% w/v) was effected by slowly adding ammonium sulphate (500 g) to the2 liters of solution and stirring for at least 2 hours after thesolution was cooled to 2° to 8° C., usually overnight. Finally theprecipitate was collected by centrifugation, dissolved in 2 L ofTris.HCl, pH 7.5, and the conductivity adjusted to approximately 3.4mS/cm by adding either ammonium sulphate (if below 3.4 mS/cm) or 10 mMTris.HCl, pH 7.5 (if higher than 3.4 mS/cm).

EXAMPLE 4

This Example illustrates the precipitation of pertactin from pertussisfermentation broth concentrates.

Ammonium sulphate (250 g/L of broth) was added to fermentor brothconcentrates and the mixture stirred for more than two hours after themixture had reached 2° to 8° C. The precipitate was collected bycentrifugation, dissolved in 10 mM Tris.HCl, pH 7.5, and the same bufferadded until the conductivity was ≦4.0 mS/cm. This solution contained allthe PT, FHA and pertactin. The solution was subjected to Perlitechromatography (as described in Example 2) to remove PT and FHA and thePerlite flow-through was subjected to hydroxyapatite and Q-Sepharose®chromatography (see Example 5).

EXAMPLE 5

This Example illustrates the chromatography of pertactin onhydroxyapatite and Q-Sepharose®.

Hydroxyapatite was packed into a suitable size column, preferably 5 cm[D]×10 cm [H] and equilibrated with 10 mM Tris.HCl buffer, pH 7.5,containing 15 mM ammonium sulphate (conductivity approximately 3.4mS/cm). Q-Sepharose® was packed into a similar column and equilibratedwith the same buffer. The two columns were connected in series with thehydroxyapatite column upstream of the Q-Sepharose® column. Theresolubilized pertactin (from Example 3) when subjected tochromatography on the two columns in series did not bind to the matricesand the flow-through fraction was collected. After filtration through a300 kDa NMWL membrane, the filtrate containing the pertactin wasconcentrated and diafiltered using ≦30 kDa NMWL membranes and finallysterile filtered using a 0.22 μm membrane.

EXAMPLE 6

This Example illustrates the purification of pertactin by binding toQ-Sepharose®.

The Perlite flow-through fraction from Example 2, was concentrated toapproximately 7 L, having a protein concentration usually between 1.5 to3.0 mg/ml. Solid ammonium sulphate was added to the concentrate at aratio of approximately 35% (w/v). The mixture was stirred for 2 or morehours at 2° to 8° C. The collected precipitate was dissolved inapproximately 500 ml of 10 mM Tris.HCl, pH 8.0, and then precipitatedwith ammonium sulphate (100 g/L). After a minimum of 2 hr. stirring at2° to 8° C., the supernatant was isolated by centrifugation. Anadditional aliquot of ammonium sulphate (100 g/L) was added to thesupernatant to precipitate the pertactin. The precipitate was dissolvedand adjusted with 10 mM Tris.HCl, pH8.0, to a conductivity ofapproximately 3.4 mS/cm. The pertactin is purified according to Example5 by passing through tandem hydroxyapatite/Q-Sepharose® columns (each 11cm [D]×8 cm [H]), ultrafiltered through a 300 kDa membrane andconcentrated with a 10 to 30 kDa membrane. The pertactin then wassolvent-exchanged by either dialysis, diafiltration or using a solventexchange column into a solution in 10 mM Tris. HCl, pH 8.0 (conductivityapproximately 0.6 mS/cm), then bound to a Q-Sepharose® column (11 cm[D]×8 cm [H]) equilibrated in 10 mM Tris.HCl, pH 8.0. The column waswashed with 10 mM Tris.HCl, pH 8.0, containing 5 mM ammonium sulphate(conductivity approximately 1.7 mS/cm) and the pertactin was eluted with10 to 100 mM (preferably 50 mM) phosphate buffer at pH 8.0. The purifiedpertactin is solvent exchanged into PBS and sterile filtered.

Alternatively, after the ammonium sulphate precipitation steps, thepertactin solution with an ionic strength adjusted to approximately 3.4mS/cm is passed through the hydroxyapatite column alone. The run-throughfraction is concentrated and solvent exchanged into 10 mM Tris.HCl, pH8.0 and bound directly onto the Q-Sepharose® column. After washing with10 mM Tris.HCl, pH 8.0, containing 5 mM ammonium sulphate, the pertactinis eluted from the Q-Sepharose® column with 10-100 mM (preferably 50 mM)phosphate buffer at pH 8.0. The purified pertactin is ultrafilteredthrough a 300 kDa membrane, concentrated and diafiltered with a 10 to 30kDa membrane and sterile filtered.

EXAMPLE 7

This Example illustrates the extraction of pertactin from B.pertussiscells.

B.pertussis cells (5% w/v) were suspended in phosphate buffered saline(10 mM sodium phosphate, pH 7.5, 0.15M sodium chloride {PBS}) containing4M urea and the suspension was dispersed for 15 seconds to 60 minutes,and stirred for 1.5 hr. at room temperature. Cell debris was removed bycentrifugation at 5 to 6,000 xg. The cellular extract was filteredthrough 100 kDa or 300 kDa membranes and the retentate diafiltered with2 to 3 volumes of PBS. The filtrate from the membranes and thediafiltrate were combined and concentrated to one-fifth the originalvolume using ≦30 kDa NWML membranes and further diafiltered with 5volumes of PBS.

EXAMPLE 8

This Example illustrates the preparation of pertactin from extracts ofB.pertussis cells,

The retentate from Example 7 was precipitated by the slow addition ofammonium sulphate (25% w/v) at room temperature and the mixture allowedto stir at 2° to 8° C. for an additional 2 hrs, preferably overnight.The precipitate was dissolved in 10 mM Tris.HCl buffer, pH 7.5, andsaturated ammonium sulphate solution added to achieve a conductivitycorresponding to that of 10 mM Tris.HCl, pH 7.5, containing 15 mMammonium sulphate (3.5 mS/cm). The pertactin was then in a form forpurification by hydroxyapatite/Q-Sepharose® chromatography as describedin Example 5.

EXAMPLE 9

This Example illustrates the immunogenicity of pertactin combined withother antigens.

A solution of purified pertactin was mixed with aluminium phosphate (3mg/ml) and varying amounts of pertactin (1 to 20 μg) were combined withconstant amounts of PT toxoid, FHA toxoid and agglutinogens. Guinea pigs(10 per group and with a 400 to 450 g weight range) were injected atdays 0 and 21 and were bled at day 28. A good antibody response topertactin was observed with doses as low as 1 μg. No significantdifference in antibody responses was observed between doses of 1 to 10μg and consistency was obtained between various lots of pertactin (SeeTable I below).

EXAMPLE 10

This Example illustrates the stability of purified pertactin.

The stability of the pertactin antigen was monitored with and withoutcombination with aluminium phosphate and after combination with otherpertussis antigens in a candidate vaccine formulation. Samples ofpertactin (without aluminium phosphate) were stored for various times at-20° C., 2° to 8° C., 24° C. and 37° C. Two lots were studied withdifferent preservatives (thimerosal and phenoxy-ethanol). It wasnoticeable that whichever preservative was used there was no reductionin the pertactin-specific ELISA value up to 3 months. A reduction wasobserved with phenoxyethanol as a preservative in the 6 and 12 monthvalues. The results are reproduced shown in Table II below.

Potential vaccine combinations in aluminium phosphate were stored at 2°to 8° C., 24° C. and 37° C. Stability of the antigen was monitored bygeneral appearance, pertactin-specific ELISA, protein content, SDS-PAGE,Western blot analysis with monospecific anti-pertactin antisera andimmunogenicity studies in guinea pigs. The pertactin has shown nochanges in stability for any storage time whether alone or incombination with either adjuvant or other antigens, as shown in TableIII below. The materials used for the experiment were pertactin alonewith aluminium phosphate adjuvant and vaccine combinations withaluminium phosphate.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides novelprocedures for recovery of pertactin in stable form suitable forincorporation as a component in a component vaccine, from fermentationproducts of Bordetella species, using column chromatography andultrafiltration. Modifications are possible within the scope of thisinvention.

                  TABLE I                                                         ______________________________________                                        DOSE RESPONSE AND CONSISTENCY                                                 OF PRODUCTION OF PERTACTIN                                                             ANTIGEN   69 kDa SPECIFIC                                                                             ANTI-69                                      LOG #    μG     ELISA μg/ml                                                                              kDa TITRES.sup.a                             ______________________________________                                        69kDa    2.0       --            9.90 ± 0.8                                69kDa    20.0      --            10.80                                                                              ± 0.7                                CP4DT001*                                                                              6.0       5.40          9.00 ± 1.20                               CP4DT003A*                                                                             6.0       6.57          9.00 ± 0.58                               CP4DT004A*                                                                             6.0       6.53          9.00 ± 1.07                               ______________________________________                                         .sup.a = Log.sub.2 (reactive titres/100): eight animals/group                 * = These materials were vaccine preparations.                                Dose: The antigens were dissolved in 1 ml and the dose/animal was 0.5 ml      on Day 0 and 0.5 ml on Day 21. Animals were bled on Day 28               

                  TABLE II                                                        ______________________________________                                        STABILITY OF UNADSORBED PERTACTIN                                                     TIME      STORAGE    PROTEIN.sup.a                                                                          ELISA                                   SAMPLE  MONTHS    TEMP (°C.)                                                                        μG/ML μG/ML                                ______________________________________                                        G2361-TH                                                                              0         --          149.sup.b                                                                             ND                                              1         6          125      143                                                       24         140      173                                                       37         136      131                                             2         6          117      169                                                       24         120      163                                                       37         119      127                                             3         6          132      156                                                       24         153      134                                                       37         128      103                                                       -20        149      160                                             6         6          117      130                                                       -20        126      104                                             12        6          126      151                                                       -20        119      131                                     G2361-P 0         --          149.sup.b                                                                             ND                                              1         6          114      127                                                       24         129      128                                                       37         125      124                                             2         6          125      121                                                       24         116      128                                                       37         132      124                                             3         6          189      126                                                       24         119      128                                                       37         147       78                                             6         6          103       94                                             12        6          117       75                                     ______________________________________                                         G2361-TH = Pertactin preparation that contains 0.01% thimerosal as the        preservative                                                                  G2361P = Pertactin preparation that contains 0.5% 2phenoxyethanol as the      preservative                                                                  .sup.a = Protein contents of the samples were determined by BCA assay         (Pierce) after TCA precipitation of the sample                                .sup.b = Protein content of sample at zero time was determined by Kjeldah

                  TABLE III                                                       ______________________________________                                        STABILITY OF PERTACTIN                                                        IN VACCINE COMBINATIONS                                                               TIME      STORAGE    ELISA  ANTI-69kDa                                LOT #   MONTHS    TEMP. (°C.)                                                                       μG/ML                                                                             TITRES.sup.a                              ______________________________________                                        A69K001P.sup.b                                                                        0         6          ND     ND                                                3         6          56     10.7 ± 1.28                                    6         6          42     9.9  ± 0.83                            A69K002P.sup.c                                                                        0         6          99     11.2 ± 1.28                                    6         6          ND     9.9  ± 1.0                             A69K003P.sup.d                                                                        0         6          58     10.9 ± 0.64                                    6         6          ND     9.5  ± 1.0                             CPDT4P.sup.e                                                                          0         6          9.44   10.1 ± 0.74                                    3         6          9.63   9.0  ± 0.00                                              24         8.43   9.0  ± 0.89                                              37         7.35   9.2  ± 0.84                                    9         6          8.19   10.5 ± 0.85                                    12        6          6.72   7.5  ± 0.93                            CP4DT001.sup.f                                                                        0         5          5.4    9.0  ± 1.20                                    3         5          6.55   10.6 ± 0.92                                    6         5          5.67   9.1  ± 1.13                            ______________________________________                                         Note:                                                                         For samples b, c and d the sample was diluted to 6 μg/ml of pertactin      prior to injection and the animals were given 0.5 ml on Day 0 and 0.5 ml      on Day 28.                                                                    Note:                                                                         For samples e and f the antigens were in 1 ml and the dose was 0.5 ml on      Day 0 and 0.5 ml on Day 28                                                    .sup.a Log.sub.2 (reactive titres/100): eight animals/group                   .sup.b Pertactin solution alone adsorbed with aluminium phosphate.            Contained 54 μg/ml of pertactin                                            .sup.c Pertactin solution alone adsorbed with aluminium phosphate.            Contained 134 μg/ml of pertactin.                                          .sup.d Pertactin solution alone adsorbed with aluminium phosphate.            Contained 77 μg/ml of pertactin.                                           .sup.e Vaccine combination containing 10 μg/ml of pertactin.               .sup.f Vaccine combination containing 6 μg/ml of pertactin.           

What we claim is:
 1. A method for the production of pertactin, whichcomprises:growing cells of Bordetella pertussis in a growth medium toprovide a culture broth containing pertactin, pertussis toxin (PT),filamentous haemagglutinin (FHA) and grown cells, separating saidculture broth from said grown cells, providing an impure aqueoussolution of pertactin substantially free from said PT and said FHA fromsaid separated culture broth, purifying pertactin in said aqueoussolution by passing said aqueous solution in contact with hydroxyapatiteand a matrix composed of an ion-exchange medium to provide unboundpertactin in flow-through and medium-bound impurities and collecting theflow-through from each said contact to provide a purified solution ofpertactin, and recovering said purified solution of pertactin.
 2. Themethod claimed in claim 1, wherein said impure aqueous solution ofpertactin is formed by:selectively removing said PT and FHA from saidculture broth, subsequently precipitating pertactin from said culturebroth, and forming said aqueous solution of pertactin by dissolving saidprecipitated pertactin.
 3. The method claimed in claim 2, wherein saidpertactin is precipitated by adding ammonium sulphate to said culturebroth.
 4. The method claimed in claim 1, wherein said impure aqueoussolution of pertactin is formed by:precipitating pertactin, PT and FHAfrom said culture broth, forming an aqueous solution of saidprecipitate, and selectively removing said PT and FHA from the resultingsolution.
 5. The method claimed in claim 4, wherein said pertactin, PTand FHA are precipitated from said growth medium by adding ammoniumsulphate to said medium.
 6. A method for the production of pertactin,which comprises:growing cells of Bordetella pertussis in a growthmedium, extracting pertactin from grown cells under non-denaturingconditions to provide an extract, subjecting the extract toultrafiltration to remove high molecular weight proteins therefrom andto provide an ultrafiltered filtrate, subjecting the filtrate to furtherultrafiltration to remove low molecular weight proteins therefrom and toprovide an ultrafiltered retentate, precipitating pertactin from saidultrafiltered retentate, forming an impure aqueous solution of pertactinsubstantially free from pertussis toxin (PT) and filamentoushaemagglutinin (FHA) by dissolving said precipitated pertactin,purifying pertactin in said aqueous solution by passing said aqueoussolution in contact with hydroxyapatite and a matrix composed of anion-exchange medium to provide unbound pertactin in flow-through andmedium-bound impurities and collecting the flow-through from each saidcontact to provide a purified solution of pertactin, and recovering saidpurified solution of pertactin.
 7. The method claimed in claim 6,wherein said ultrafiltration of said extract is effected using about 100to 1000 kDa Nominal Molecular Weight Limit (NMWL) membrane.
 8. Themethod of claim 7, wherein said filtrate is diafiltered using an about30 kDa or less NMWL membrane prior to said precipitation.
 9. The methodclaimed in claim 6, wherein said pertactin is precipitated by addingammonium sulphate to said ultrafiltered retentate.
 10. The method ofclaim 3, wherein said precipitated pertactin is dissolved in a low ionicstrength buffer solution at a pH of about 6.0 to about 8.5.
 11. Themethod of claim 10 wherein said buffer solution has an ionic strength ofless than about 4 mS/cm.
 12. The method claimed in claim 1 or 6 whereinsaid purified solution is further subjected to ultrafiltration to removepyrogens using a membrane of about 100 to 300 kDa Nominal MolecularWeight Limit (NMWL).
 13. The method claimed in claim 12, wherein saidultrafiltered purified solution is further concentrated using a membraneof about 30 kDa or less NMWL.
 14. The method of claim 6 wherein saidprecipitated pertactin is dissolved in a low ionic strength buffersolution at a pH of about 6.0 to about 8.5.
 15. The method of claim 14wherein said buffer solution has an ionic strength of less than about 4mS/cm.
 16. The method of claim 12 wherein said ultrafiltered pertactinsolution is passed in contact with an ion-exchange matrix to bindpertactin thereto and purified pertactin is selectively eluted from theion-exchange medium.
 17. A method for the production of pertactin, whichcomprises:growing cells of Bordetella pertussis in a growth medium toprovide a culture broth containing pertactin, pertussis toxin (PT),filamentous haemagglutinin (FHA) and grown cells, separating saidculture broth from said grown cells, providing an impure aqueoussolution of pertactin substantially free from said PT and said FHA fromsaid separated culture broth, purifying pertactin in said aqueoussolution by passing said aqueous solution in contact with hydroxyapatiteat a conductivity of less than 1.5 mS/cm to bind said pertactin to saidhydroxyapatite and/or a matrix composed of an ion-exchange medium at aconductivity of less than 2.8 mS/cm to bind said pertactin to saidmatrix, thereafter selectively eluting said bound pertactin fromhydroxyapatite and/or said matrix with a buffer having a conductivity ofgreater than 1.5 mS/cm for hydroxyapatite and greater than 2.8 mS/cm forsaid ion-exchange medium, and collecting flow-through from each saidelution to provide a purified solution of pertactin, and recovering saidpurified solution of pertactin.